Vascular stem cells and progenitors

Vascular stem cells and progenitors

Lipnik Karoline

Department of Vascular Biology and Thrombosis Research

Medical University of Vienna

Lazarettgasse 19

A-1090 Vienna

Austria

Basic seminar 1, Vascular Biology, N090/N094, SS 2008

16. June 2008

Overview

Development of blood vessel system – vasculogenesis, angiogenesis

Embryonic stem cells Alternatives to embryonic stem cells Adult stem and progenitor cells and

prospective therapeutical applications

Vascular development – the beginningcygote blastocyste

Germ layers give rise to development of defined cell types

http://www.hhmi.org/biointeractive/stemcells/animations.html

ENDODERM

Lungs

Liver

Pancreas

Intestine

Molecular mechanisms of stem-cell identity and fateFiona M. Watt and Kevin Eggan

EKTODERM

Brain Skin, Hair

Mammary gland


MESODERM – VASCULAR SYSTEM

Haematopoiesis

MesenchymeMuscle

Heart

Vascular cells


Blood vessel formation

Two categories:

a.) vasculogenesis:

de novo blood vessel generation from vascular progenitor cells

b.) angiogenesis:

formation of new blood vessels via extension or remodeling of existing blood vessels;

Blood vessel formation

Vasculogenesis:

a.) during embryonic development;

b.) during adulthood associated with circulating progenitor cells

Angiogenesis:

a.) embryonic development

b.) adulthood: wound healing, menstrual cycle, tumour-angiogenesis…

Vasculogenesis

The vascular system is one of the earliest organ system that developes during embryogenesis

One of the first markers of angioblast precursors Flk-1 (VEGF-R2)

Further important early markers are: Brachyury and C-Kit

Vasculogenesis

1. First phase• Initiated from the generation of hemangioblasts; leave

the primitive streak in the posterior region of the embryo; a part of splanchnic mesoderm

2. Second phase• Angioblasts proliferate and differentiate into

endothelial cells

3. Third phase• Endothelial cells form primary capillary plexus

Vasculogenesis

Extraembryonic Vasculogenesis

Intraembryonic Vasculogenesis


First apparent as blood islands in yolk sac Blood islands are foci of hemangioblasts Differentiate in situ:

a.) loose inner mass of embryonic hematopoietic precursors

b.) outer layer of angioblasts by the merge of individual blood islands capillary

networks are formed Yolk sac vasculogenesis communicate with fetal

circulation via the vitelline vein


Cellular composition of the yolk sac

yolk sac

bloodisland

endodermendothelial cell

blood

mesoderm

Human yolk sac with blood island

endoderm

mesoderm

Blood island

ec

bloodislandse

ac

aleem

te

bl

7.5 days pcblood islands

8.5 days pc 12.5 days pcvascular vitelline

circulationchannels

lacZ

ε-globinHuman

+1

proβ-LCR

ε- - globin lacZ transgenic mice

Intraembryonic vasculogenesis

para-aortic mesoderm =

AGM (aorta-gonad-mesonephros) First dorsal aorta and

cardinal veins are built Endocardium - vascular plexus is generated Development of bilateral embryonic aortae Then allantoic vasculature occurs

Embryonic circulatory system

Intraembryonic vasculogenesis

Subsequent vascular development primarly via angiogenesis

Some endoderm derived organs, however, are also capable for vasculogenesis

Developmental angiogenesis

Majority of vascular development occurs via angiogenesis

Growth of new blood vessels from existing vessels

Two distinct mechanisms available

a.) sprouting angiogenesis

b.) intussusceptive angiogenesis

Sprouting angiogenesis

Sprouting: invasion of new capillaries into unvascularized tissue from existing mature vasculature

- degradation of matrix proteins

- detachment and migration of ECs

- proliferation

Guided by endothelial tip cells and influenced by various attractant and repulsive factors (Ephrin, Netrin, Plexin…)

Sprouting angiogenesis

Intussusceptive angiogenesis

Intussusceptive or non sprouting angiogenesis:

- remodelling of excisting vessels

- vessel enlarges

- pinches inward

- splits into two vessels

Intussusceptive angiogenesis

Das Endothel ein multifunktonelles Organ: Entdeckung, Funktionen und molekulare Regulation

Stürzl M., et al Cell Tissue Res (2003) 314:107–117DOI 10.1007/s00441-003-0784-3

Building of blood vessels in adulthood

Endothelial precursors

Intussusceptive growth Angiogenic sprouting

Important factors guiding angiogenesis - VEGF

Important factors guiding angiogenesis

• bFGF: proliferation, differentiation, maturation

• TGFβ: stabilize the mature capillary network by

strengthen the ECM structures

• PDGF: recruits the pericytes to provide the

mechanical flexibility to the capillary

• MMP inhibitors: suppresses angiogenesis

• Endostatin: cleaved C-terminal fragment of collagen

XVII; binds to VEGF to interfere the binding to VEGFR

Summary part 1 Vascular system developes from mesodermal germ layer Two categories of vessel building:

a.) vasculogenesis: vascular progenitors

b.) angiogenesis: sprouting, intussusceptive; from preexisting vessels

Extraembryonic vasculogenesis: yolk sac, blood islands, vascular plexus

Intraembryonic vasculogenesis: AGM region – dorsal aorta and cardinal veins

Majority of blood vessels built by angiogenesis (embryo and adult) Proangiogenic factors: VEGF, bFGF, angiopoietins Maturation and stabilization: TGFß and PDGF Anti-angiogenic: MMP inhibitors, Endostatin

Embryonic stem cells as a tool to study vascular development

generation of stem cells Differentiation to various cell types


Embryonic stem cells

Generation


Embryonic stem cells as a tool to study vascular development

Characteristics derived from blastocyst: 3-5 day-old embryo Unspecialized - totipotent potential to develop all different cell types Divide without limit: long term self renewal

Tests to identify embryonic stem cellsA. Subculturing for many monthsB. Specific surface markers: Oct-4, Sox2,

NANOGC. Testing if cells are pluripotent: differentiation

in cell culture;D. injecting in vivo - teratoma should be built

Differentiation of stem cells to vascular cells

Differentiation of ES cells to vascular cells

Stem cells cultivated with a defined cocktail mix (BMP-4, VEGF, SCF, Tpo, Flt3-ligand) in serum free medium to generate embryoid bodies

EBs dissociated and cultivated in specific medium or EBs seeded for outgrowth

KDRlow/C-Kitneg population gives rise to cardiomyocytes, SMCs and Ecs – common progenitor

Lei Yang et al., Nature Letters, 2008

Generation of functional hemangioblasts from embryonic stem cells

LDL – redvWF - green

LDL – redVE-cad- green

CD31– redvWF - green

Summary part 2

Embryonic stem cells are generated from the inner cell mass of blastocystes (3 – 5 dpc)

Characteristics: indefinite life span, totipotent – can give rise to every cell type

Primarly cultivated on feeder cells for expansion of undifferentiated cells

Generation of ECs through stimulation with various cytokine cocktail – Embryoid bodies, outgrowth

Alternatives to human embryonic stem cells

Stem cells derived from single blastomeres

Stem cells through nuclear reprogramming – overview

Induced pluripotent stem cells (iPS) through expression of stem cell specific proteins in differentiated cells

Human embryonic cell lines derived from single blastomeres

endoderm

alpha-fetoprotein

mesoderm

SMC

ectoderm

3-tubulin

Figure 1. Derivation and Characterization of hESC Lines from Single Blastomeres without Embryo Destruction(A) Stages of derivation of hES cells from single blastomere. (a) Blastomere biopsy, (b) biopsied blastomere (arrow) and parent embryo are developing next to each other, (c) initial outgrowth of single blastomere on MEFs, 6 days, and (d) colony of single blastomere-derived hES cells.

Stem cells through nuclear reprogramming - overview

Adult and stem cells are genetically equivalent Differential gene expression is a result of epigenetic

changes during development Nuclear reprogramming: reversal of the

differentiation state of a mature cell to one that is characteristic of the undifferentiated embryonic state

A. Nuclear transfer

B. Cellular fusion

C. Cell extracts

D. Culture induced reprogramming


Experimental Approach:Reproductive cloning:functional test for reprogramming to totipotencySomatic cell nuclear transfer:efficient derivation of genetically matched ES cells with normal potency

Mechanistic insights:Allows epigenetic changes (reversible) to be distinguishedfrom genetic changes (irreversible)

Nuclear transfer

Limitations:Reproductive cloning is very inefficientThere are abnormalities at all stages ofdevelopment

Nuclear exchange to generate stem cells


Cybrid embryos - human chromosomes with animal eggs

In vitro fertilization

Intracytoplasmic sperm injection

Somatic cell nuclear transfer


Cell fusion

Experimental Approach:Nuclear reprogramming of somatic genome in hybrids generated with pluripotent cells; in most hybrids less differentiated partner is predominant

Mechanistic insights:Allows study of genetics of reprogrammingQuestion: chromosomes of somatic cells reprogrammed or silenced; nucleus or cytoblast required for molecular reprogramming

Limitations:Fusion rate is very lowTetraploid cells are generated


Cell extract

Experimental Approach:Exposure of somatic nuclei or permeabilized cells to extracts from oocytes or pluripotent cells;

Mechanistic insights:Allows biochemical and kinetic analysis of reprogramming

Limitations:No functional reprogramming done


Cell explantation

Experimental Approach:Explantation in culture selects for pluripotent, reprogrammed cells; certain physiological conditions entire cells can de-differentiate (Teratokarzinoma)

Mechanistic insights:Allows study of genetics of reprogramming

Limitations:May be limited to germ line cells

Stem cells through nuclear reprogramming - overview Molecular mediators of reprogramming and

pluripotency Chromatin remodelling factors DNA modification Histone modification Pluripotency maintained by a combination of extra-

and intracellular signals Extracellular signals: STAT3, BMP, WNT Intracellular signals: factors at transcriptional level

(Oct-4, Nanog, Sox2…)


Downstream targets: transcription factors, which are silent in undifferentiated cells

Polycomb group (PcG) proteins – chromatin modifiers – repress developmental pathways

Chromatin formation of many key developmental genes: bivalent domains

Activating and inhibitory marks Bivalent domains are lost in differentiated cells

Induced pluripotent stem cells (iPS) and cellular alchemy

introducing of factors in fibroblasts - induced pluripotent stem cells Able to produce many cell types Initially 24 genes selected Transduced into mouse embryonic fibroblasts Resistance gene for G418 under control of Fbx15 promoter, which is

only active in pluripotent cells Drug resistant colonies appeared, which resembled ES cells Expressed transcripts and proteins considered to be part of ES cell

signature Termed: induced pluripotent stem cells (iPS) Formed all three germ layers in vitro and in vivo Best combination: Oct-4, Sox2. c-Myc, Klf4

Summary part 3

Generation of ESCs from single blastomere Reprogramming of differentiated cells via:

- nuclear transfer: molecular cloning

- transduction with stem cell genes

Adult progenitor and stem cells and potential clinical application

Undifferentiated cells found among differentiated ones

Identified in various tissues mainly generate cell types of the tissue in

which they reside can renew themselves (20 to 30 PD) Can differentiate Task: Maintenance and repair

Adult progenitor and stem cells – sources and transdifferentiation

Adult stem cells

Adult progenitor cells

Mobilization of vascular progenitors

Tissue ischemia results in expression of cytokines like VEGF

Recruites progenitor cells Steady state 0.01% MNCs in blood are CEPs Amount of circulating progenitors are

increased after trauma, infectious injuries or tumour growth

24 h after injury: 12%

Mobilization of vascular progenitors

Mobilization mediated through: metalloproteinases, adhesion molecules, VEGF and PLGF (placental growth factor)

Induction of MMP9 causes release of stem-cell active soluble kit ligand

Isolation of adult vascular progenitors

Prospective therapeutical applications

Tumour homing – Trojan horse principle Organ revascularization and regeneration Wound healing Heart diseases Blood diseases

Tumour homing – Trojan horse principle

Home to places of active neoangiogenesis

Vascular progenitor transduced with a therapeutic gene

Vehicle for targeting therapeutic gene expression to tumour

Bystander effect of advantage

Gene directed enzyme prodrug therapy(CYT-P450-Ifosfamide; HSV-TK/Ganciclovir)

Organ vascularization and regeneration

After pathological ischemic events in the body exogenous introduction of vascular progenitors may facilitate restoration

Bone marrow, rich reservoir of tissue-specific stem and progenitor cells

Possible applications: ischemic limbs, postmyocardial infarction, endothelialization of vasclular grafts, atherosclerosis, retinal and lymphoid organ neovascularization

Potential use of adult stem and progenitor cells

Peripheral Artery Disease

Figure 5. ﾊ Angiographic analysis of collateral vessel formation in patients in group A Collateral branches were strikingly increased at (A) knee and upper-tibia and (B) lower-tibia, ankle, and foot before and 24 weeks after marrow implantation. Contrast densities in suprafemoral, posterior-tibial, and dorsal pedal arteries (arrows) are similar before and after implantation.

Tateishi-Yuyuama et al., The Lancet, Aug, 2002

Impaired wound healing

Figure 4.Limb salvage after marrow implantation in two patients in group ANon-healing ulcer on heel (A) and ischaemic necrosis on big toe (B) showed improvement 8 weeks after implantation.

Tateishi-Yuyuama et al., The Lancet, Aug, 2002

Summary part 4

Adult progenitors are undifferentiated cells with high capacity to proliferate

Can be found in many different tissues Differentiate to resident cell types Transdifferentiation Can be isolated by sorting for progenitor markes,

differentiated and used for clinical applications Various potential applications Tissue vascularization and regeneration, heart diseases,

… Ongoing preclinical and clinical trials

Literature IEndothelial BiomedicineWilliam C. AIRDCambridge University Press, 2007

Yolk Sac with Blood islandsNew England Journal of medicineVolume 340:617, February 1999

Developmental Biology, 8th EditionSinuauer Associates, 2006

Hematopoietic induction and respecification of A-P identity by visceral endoderm signaling in the mouse embryoMaria Belaoussoff et al.Development, November 1998

Human cardiovascular progenitor cells develop forom a KDR+ embryonic-stem-cell-derived populationLei Yang et al.Nature Letters, May 2008

Generation of functional hemangioblasts from human embryonic stem cellsShi-Jiang Lu et al.Nature Methods, May 2007

Human embryonic stem cell lines derived from single blastomeresIrinia Klimanskaya et al.Nature Letters, November 2006

Literature IINuclear Reprogramming and pluripotency

Konrad Hochedlinger et al.

Nature, June 2006

Human-animal cytoplasmic hybrid embryos, mitochondra, and an energetic debate

Justin St Jon et al.

Nature Cell Biology, September 2007

Induced pluripotency and cellular alchemy

Anthony C F Perry

Nature Biotechnology, November 2006

Endothelial progenitor cells for cancer gene therapy

K-M Debatin et al.

Gene Therapy, 2008

Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration

Shahin Rafii et al.

Nature Medicine, June 2003

Documents

Vascular stem cells and progenitors